Correspondence: Lalit C. Garg, Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: +91 11 2670 3652; fax: +91 11 2674 2125; e-mail: email@example.com
Sequencing analysis of the complete genome of Mycobacterium tuberculosis (Mtb) H37Rv resulted in the identification of a novel multigene, the PE family of genes. The genes of the largest PE_PGRS subfamily of the PE family are mainly restricted to pathogenic mycobacteria, and their exact role in the biology of Mtb is not clearly understood. Based on their sequence homology, PE_PGRS proteins were initially thought to serve common functions. However, studies on individual proteins reveal that the individual proteins of this subfamily could be performing several unrelated tasks. In the present study, we investigated the function of PE_PGRS30 by expressing it in Mycobacterium smegmatis. PE_PGRS30 expression in M. smegmatis resulted in phenotypic changes with altered colony morphology and growth profile. The recombinant PE_PGRS30 showed polar localization and was found to be associated with the cell wall of M. smegmatis. Thus, the present study suggests that the prolonged lag phase of growth caused by the PE_PGRS30 may, in part, contribute to the latency of Mtb.
The success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis as a pathogen, is attributed to its slow growth and its ability to cause latent infection, which later turns into an active infection when host immunity weakens (Parrish et al., 1998). A true understanding of the biology of a pathogen is essential for the successful control of the disease. Sequencing analysis of the complete genome of Mtb H37Rv revealed the existence of two novel, multigene families, the PE family and the PPE family, accounting for ∼5% of the total coding capacity of the Mtb genome. The members of this gene family have been found to be present only in pathogenic mycobacteria (Singh et al., 2008). The genes of the PE family are characterized by a conserved amino-terminal domain (PE domain) with proline and glutamic acid residues at positions 8 and 9, respectively (Cole et al., 1998). Based on the domain composition, PE genes can be categorized into three classes, the largest class of which is the PE_PGRS subfamily, consisting of 61 members.
The PE_PGRS (proline-glutamic acid_polymorphic GC-rich repetitive sequence) family contains genes in which the PE domain is linked at the C-terminus with a highly variable Gly-Ala-rich sequence (PGRS domain) (Lamichhane et al., 2003). The presence of a large number of homologous genes belonging to the PE_PGRS subfamily in the genome of pathogenic mycobacteria raises anticipation regarding the significance of these genes in the functionality of the organism.
The exact function for the majority of these proteins, present mainly in pathogenic mycobacteria, has not yet been elucidated (Brennan et al., 2004). Variable expression of some PE_PGRS genes has been observed under conditions mimicking infection, thus implicating a possible role for these proteins in mycobacterial pathogenesis (Saviola et al., 2003; Dheenadhayalan et al., 2006b). PE_PGRS30, one of the members of the PE_PGRS subfamily, is upregulated during Mtb infection of bone-marrow-derived macrophages (Delogu et al., 2006). These findings indicate that there is a need to decipher the functions of individual PE_PGRS proteins.
Therefore, the present study was envisaged to decipher the precise role of PE_PGRS30 in the pathogenesis of Mtb by examining its effect on Mycobacterium smegmatis, a fast-growing mycobacterial species that naturally lacks this protein. For this purpose, the gene for PE_PGRS30 (Rv1651c) was cloned in Escherichia coli/Mycobacterium shuttle vector and introduced into M. smegmatis. The results illustrate that PE_PGRS30 modulates the growth of M. smegmatis. The present data demonstrate for the first time the effect of any PE_PGRS protein on the growth of Mycobacterium.
Materials and methods
Construction of vectors and recombinants
The Rv1651c gene of Mtb H37Rv, amplified using the M. tuberculosis Bacterial Artificial Chromosome DNA library as a template (Brennan et al., 2004) and gene-specific primers (forward with NdeI site – 5′-CCCCATATGTCGTTCTTACTCGTGGAGCC-3′; reverse with HindIII site – 5′-AAGCTTAGGGGCAATTGCTGCGC-3′), was cloned into pGEMT-easy vector (Promega, Madison, WI). The NdeI–HindIII-digested PCR product was then cloned downstream to the heat shock protein 60 (hsp60) promoter of the E. coli/Mycobacterium shuttle plasmid, pVV16 (Stover et al., 1991) to generate the plasmid pVV1651c. To create a GFP-PE-PGRS30 fusion product, the green fluorescent protein (GFP) gene amplified from pGFP plasmid (accession no. U17997), using the forward and reverse primers with HindIII and ClaI sites, respectively (5′-AAGCTTATGAGTAAAGGAGAAGAAC-3′; 5′-ATCGATTTACTATTTGTATAGTTCATCCATGCC-3′), was cloned in pGEMT-easy. The GFP gene released by HindIII and ClaI digestion was inserted into pVV16 either alone or in fusion at the 3′-end of Rv1651c using HindIII and ClaI sites to generate the recombinant constructs, pVVGFP and pVV1651c−GFP, respectively.
Microorganisms, media and transformation
Escherichia coli DH5α cells were grown in Luria–Bertani (LB) broth and LB agar (Difco Laboratories) with appropriate antibiotics, at 37 °C. Mycobacterium smegmatis mc2155 cells were grown in liquid medium 7H9 supplemented with Albumin–Dextrose–Catalase (ADC) enrichment (Difco Laboratories) and Tween 80 (0.05%). Cell preparation and electroporation were carried out using standard protocols (Parish & Stroker, 2008). Transformants were selected on 7H11 solid medium supplemented with oleic acid-ADC enrichment (Difco Laboratories) containing 100 μg mL−1 hygromycin and 25 μg mL−1 kanamycin (Sigma-Aldrich).
Cell fractionation and immunoblot analysis
Mycobacterium smegmatis cell fractionation was carried out essentially as described earlier, with minor modifications (Delogu et al., 2004). Briefly, the recombinants grown up to the late log phase were harvested by centrifugation at 3000 g for 10 min at 4 °C, followed by washing with cold phosphate-buffered saline (PBS) and finally sonication in PBS containing the protease inhibitor P-8849 (Sigma-Aldrich). The whole-cell lysate thus prepared was centrifuged at 20 000 g to separate the insoluble (pellet) and the soluble (supernatant) fractions.
Samples were subjected to SDS-PAGE as described by Laemmli (1970) and subjected to Western blot analysis essentially as described earlier (Alone et al., 2007) using anti-GFP monoclonal antibody (Roche, Germay). The blot was developed with a horseradish peroxidase-labeled anti-mouse IgG antibody (Sigma-Aldrich) and a chemiluminescent substrate system (Biological Industries, Israel).
Mycobacterium smegmatis cells were allowed to grow at 200 r.p.m. at 37 °C. OD600 nm was measured every 3 h using a Perkin-Elmer spectrophotometer. To analyze the growth kinetics, OD600 nm was plotted against time on a semi-log plot.
Wild-type and transformed M. smegmatis were fixed with 4% paraformaldehyde and coated on poly-lysine treated coverslips, which were then mounted on slides using vectashield mounting medium (Vector Laboratories Inc.). Microscopic visualization was performed on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) using an oil immersion objective.
Immunoelectron microscopy of M. smegmatis cells was performed essentially as described earlier (Burghardt & Droleskey, 2006) at the Electron Microscopic Facility, Advanced Instrument Research Facility, JNU, New Delhi. Briefly, M. smegmatis cells from log-phase cultures were fixed with 4% paraformaldehyde containing 0.5% glutaraldehyde and concentrated in 2% agar. The agar-encased bacteria were then dehydrated and embedded using LR white resin (Electron Microscopy Sciences). Thin sections (100 nm) were obtained using Leica Ultracut (Leica, Germany) and processed for immunostaining using anti-GFP antibody and gold (10 nm)-labeled anti-mouse secondary antibody. Unrelated antibody was used at a similar dilution as a negative control. The immunostained sections were viewed using a Jeol 2100F transmission electron microscope (Jeol Analytic Instruments).
Expression of PE_PGRS30 alters the colony morphology of M. smegmatis
The colonies of the M. smegmatis transformed with pVV1651cGFP (pVV1651cGFPMs) appeared on the solid agar-based medium after 4 days of plating, while the colonies transformed with vector alone (pVVGFPMs) appeared within 3 days. On day 5, PE_PGRS30-transformed M. smegmatis colonies were smaller in size when compared with the control (Fig. 1a and b).
Effect of PE_PGRS30 on the growth kinetics of M. smegmatis
The M. smegmatis cultures transformed with the pVV1651c showed a significant lag in growth when compared with that transformed with the pVV16. While the vector-transformed M. smegmatis cells exhibited a uniform growth rate till the cell culture reached the stationary phase of growth, the pVV1651c transformants showed growth retardation at 12 h, with a resumption of normal growth rate after 30 h, as shown in Fig. 1c. The doubling time calculated for the pVV1651c-transformed M. smegmatis (∼8.91 h) was significantly higher than that of the M. smegmatis cells transformed with the control plasmid (∼5.81 h), as established from the growth curve. The numbers of CFU formed upon saturation by these two strains were found to be equal and the majority of the cells (>70%) were expressing the recombinant Rv1651c.GFP fusion protein. These data suggest that resumption to the same log-phase growth rate is not due to nonexpressing M. smegmatis cells following antibiotic consumption.
Expression and localization analysis of PE_PGRS30 in a mycobacterial cell
In order to study the expression of PE_PGRS30 in M. smegmatis, the expression of C-terminal GFP fusion of PE_PGRS30 was analyzed by immunoblotting with anti-GFP antibody (Fig. 2a). The analysis revealed that the PE_PGRS30-GFP did not express as one intact protein as multiple bands (∼70–120 kDa) appeared on the blot.
Fluorescence microscopy demonstrated that the GFP fluorescence in the pVV1651cGFPM. smegmatis recombinants was not dispersed throughout the cell, but was confined to either one or both the poles of the cell (Fig. 2b). In contrast, pVVGFPM. smegmatis transformants showed uniform fluorescence throughout the cell, without being confined to a specific location.
Immunoblot analysis of the subcellular fractions of the pVV1651cGFPM. smegmatis recombinants revealed that all the cleavage products of PE_PGRS30-GFP were localized in the insoluble fraction of the cell preparation (Fig. 2a, bottom panel). On the contrary, GFP protein expressed by the pVVGFP recombinant strain was present in the soluble fraction (Fig. 2a).
Localization of PE_PGRS30-GFP fusion protein was studied by immunoelectron microscopy of the pVV1651cGFP and pVVGFPM. smegmatis recombinants. The expression of GFP in pVV1651cGFP was exclusively associated with the cell wall, whereas it exhibited cytoplasmic localization in the pVVGFPM. smegmatis transformants (Fig. 3). Immunolabeling using an unrelated primary antibody did not show any staining, indicating the specificity of the staining procedure.
Mtb is an extraordinary pathogen that can reside in host macrophages for decades without replicating. However, the exact mechanism of nonreplicating persistence, the genes and factors responsible for this state, and its reversal are not clearly understood. A possible approach to address this problem is to study the unique features of the Mtb genome, one of them being the genes of the PE_PGRS subfamily.
Functions of the mycobacterial proteins are often studied by expressing the genes from virulent strains in nonvirulent mycobacteria and monitoring the bacteria for gain of function (Cosma et al., 2003; Huang et al., 2010).
Despite the high degree of homology among the members of the PE_PGRS subfamily, and the general notion of them being present as cell-surface antigens and responsible for antigenic variability, some PE_PGRS proteins have also been implicated in entirely different functions (Deb et al., 2006; Tian & Jian-Ping, 2010 and references there in). For example, PE_PGRS62 has been reported to downregulate the inflammatory response by decreasing the expression of interleukin-1β (IL-1β) and IL-6 (Huang et al., 2010), whereas PE_PGRS33 expression resulted in necrosis or apoptosis of macrophages, upregulated LDH and IL-10 and reduced NO and IL-12 levels (Dheenadhayalan et al., 2006a).
Significant phenotypic changes were observed in the pVV1651c-transformed M. smegmatis expressing the PE_PGRS30 protein. The change in the morphology and size was not due to the change in cell wall composition as no significant differences in the sensitivity to antibiotics (rifampin, streptomycin and ethambutol) or detergent (SDS) were observed between the vector-transformed and pVV1651c-transformed M. smegmatis cells (data not shown). Also, no detectable differences in the protein profile of the two were noticed on SDS-PAGE (data not shown). Electron microscopy of intact bacteria also revealed that the difference in colony morphology was not due to altered bacterial cell structure, as observed with PE_PGRS33 (Delogu et al., 2004). The unusual growth pattern observed in the pVV1651cM. smegmatis transformants was similar to that of M. smegmatis transformed with α-crystallin-like small heat shock protein (Yuan et al., 1996). This protein, present only in slow-growing mycobacteria, is thought to be involved in protein stability and the long-term survival of Mtb during latent infections (Yuan et al., 1996). Because the members of the PE-PGRS family share a huge degree of homology, a few other PE_PGRS proteins viz. PE_PGRS16, PE_PGRS26 and PE_PGRS62 were tested for their effect on M. smegmatis growth. However, no change in the growth pattern was observed with these, suggesting that the retardation in the growth is PE_PGRS30 specific.
Mycobacteria are known to show polar growth, where cell wall synthesis material and machinery are targeted to the tips of the bacterium (Thanky et al., 2007). Polar localization of the PE_PGRS30-GFP fusion protein in M. smegmatis suggests that PE_PGRS30 inhibits growth directly or indirectly by regulating cell wall synthesis. Further insight into the localization of PE_PGRS30 by subcellular fractionation and immuno-electron microscopy showed it to be present in the cell wall of Mycobacterium. Cytoplasmic localization and detection of the GFP in the soluble fractions only in the pVVGFP-transformed M. smegmatis confirms the integrity of the cellular fractions and the authenticity of the immunoelectron microscopy. Different forms of PE_PGRS30-GFP fusion protein detected in Western blots might be the truncated forms of the protein resulting from cleavage at various sites. Western blot using antibodies against the N-terminal domain, PE domain (amino acids 1–132), of PE_PGRS30, also generated a similar pattern of bands (data not shown). Thus, from the present data, it is not clear whether the cleavage occurs from the N-terminus or the C-terminus.
Thus, the present study, together with the reports of Ramakrishnan et al. (2000), indicates a possible role of PE_PGRS30 in latency of the Mtb. Insights into the mechanism of growth retardation brought about by PE_PGRS30 and studies using animal models will determine the precise role of this protein in the biology of Mtb, which will aid in the development of more potent vaccines and drugs against the pathogen.
The Department of Biotechnology, New Delhi, is acknowledged for financial support. The Council of Scientific and Industrial Research, New Delhi, is acknowledged for research fellowship to V.K.G. The authors sincerely appreciate the technical help provided by Mr S.C.P. Sharma and Dr Gajender Saini at the Advanced Instrumentation Research Facility (AIRF), JNU, New Delhi, for electron microscopy.